Galactose-Decorated Reduction-Sensitive Degradable Chimaeric

Jul 1, 2013 - Confocal microscopy showed that FITC-CC-loaded Gal-decorated ... These reduction-responsive chimaeric biodegradable polymersomes offer ...
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Galactose-Decorated Reduction-Sensitive Degradable Chimaeric Polymersomes as a Multifunctional Nanocarrier To Efficiently Chaperone Apoptotic Proteins into Hepatoma Cells Xiaoyan Wang, Huanli Sun, Fenghua Meng,* Ru Cheng, Chao Deng, and Zhiyuan Zhong* Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China S Supporting Information *

ABSTRACT: Hepatoma-targeting reduction-sensitive chimaeric biodegradable polymersomes were designed and developed based on galactose−poly(ethylene glycol)−poly(ε-caprolactone) (Gal-PEG-PCL), PEG−PCL−poly(2-(diethylamino)ethyl methacrylate) (PEG-PCL-PDEA, asymmetric), and PEG-SS-PCL for facile loading and triggered intracellular delivery of proteins. The chimaeric polymersomes formed from PEG-PCL-PDEA and PEG-SS-PCL had a monodisperse distribution with average sizes ranging from 95.5 to 199.2 nm depending on PEG-SS-PCL contents. Notably, these polymersomes displayed decent loading of bovine serum albumin (BSA), ovalbumin (OVA), and cytochrome C (CC) proteins likely due to presence of electrostatic and hydrogen bonding interactions between proteins and PDEA block located in the interior of polymersomes. The in vitro release studies showed that protein release was largely accelerated under a reductive condition containing 10 mM dithiothreitol (DTT). For example, ca. 77.2 and 22.1% of FITC-BSA were released from CP(SS50) (chimaeric polymersomes containing 50 wt % PEG-SS-PCL) at 37 °C in 12 h in the presence and absence of 10 mM DTT, respectively. Confocal microscopy showed that FITC-CC-loaded Gal-decorated CP(SS40) could efficiently deliver and release FITC-CC into HepG2 cells following 24 h treatment, in contrast to little or negligible fluorescence detected in HepG2 cells treated with FITC-CC-loaded nontargeting polymersomes or free CC. MTT assays revealed that CC-loaded Gal-decorated CP(SS40) exhibited apparent targetability and pronounced antitumor activity to HepG2 cells, in which cell viabilities decreased from 81.9, 60.6, 49.5, 42.2 to 31.5% with increasing Gal-PEG-PCL contents from 0, 10, 20, 30 to 40 wt %. Most remarkably, granzyme B-loaded Gal-decorated chimaeric polymersomes effectively caused apoptosis of HepG2 cells with a markedly low half-maximal inhibitory concentration (IC50) of 2.7 nM. These reductionresponsive chimaeric biodegradable polymersomes offer a multifunctional platform for efficient intracellular protein delivery.

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INTRODUCTION Protein drugs have emerged as potent medicines for various human diseases including diabetes and cancers.1−3 As compared to chemotherapeutics, protein drugs have unique advantages of high specificity, superior anticancer efficacy, and low side effects. The application of protein drugs, however, encounters several challenges including rapid degradation and elimination following iv injection, poor bioavailability, low cell permeability, and inferior intracellular trafficking.3 In the past years, different nanocarriers such as liposomes,4 polymeric nanoparticles,5−7 and polymersomes8−10 have been investigated for intracellular protein delivery. In particular, nanosized polymersomes self-assembled from amphiphilic block copolymers have appeared most ideal in that they present a watery core for loading of proteins, have better stability as compared to liposomes, and are intrinsically stealthed by nonfouling polymers.11−14 For example, Palmer et al. reported that hemoglobin-loaded PEG−poly(ε-caprolactone) (PEG-PCL) © 2013 American Chemical Society

polymersomes had similar oxygen affinities to human red blood cells.15 Kataoka et al. developed myoglobin-encapsulated polyion complex vesicles which could perform reversible oxygenation/deoxygenation reaction.16 Discher et al. explored encapsulation of insulin into PEG-PBD polymersomes.17 These polymersomes, however, usually exhibit low protein loading capacity, poor cellular uptake, and/or slow protein release inside target cells. In the past years, stimuli-sensitive polymersomes that release payloads in response to an internal or external stimulus have been developed for programmed protein and drug delivery.18−27 For example, pH-sensitive degradable polymersomes were designed for active intracellular release of anticancer drugs,28 monosaccharide-responsive polymersomes for conReceived: May 20, 2013 Revised: June 29, 2013 Published: July 1, 2013 2873

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Scheme 1. Schematic Illustration on Hepatoma-Targeting Reduction-Sensitive Biodegradable Chimaeric Polymersomes for Active Loading and Intracellular Release of Proteins

trolled insulin release,29 and photosensitive polymersomes for triggered release of proteins.30 In particular, reduction-sensitive polymersomes have received much interest for active intracellular protein release due to presence of a high reducing potential in the cytosol and cell nucleus.18,19,31 For instance, Hubbell et al. reported that reduction-sensitive PEG−SS− poly(propylene sulfide) polymersomes delivered and released calcein to cells in 10 min.32 We found that dual-sensitive polymersomes based on PEG-SS-PDEA33 and PEG-PAAPNIPAM34 could efficiently load and release exogenous proteins into cells leading to improved cell apoptosis. The cellular uptake of polymersomes could be enhanced by installing specific targeting ligands such as antibody, peptide, or lactoferrin.35−37 For instance, Feijen et al. reported that antiEGFR decorated enzyme-sensitive degradable polymersomes efficiently delivered FITC-dextran into SKBR3 cells.38 In order to enhance protein and drug loading levels, chimaeric polymersomes that present an ionic interior for active interaction with cargoes have been developed based on asymmetric triblock copolymers.9,39 In this paper, we report on hepatoma-targeting, reductionsensitive, chimaeric biodegradable polymersomes based on galactose−poly(ethylene glycol)−poly(ε-caprolactone) (GalPEG-PCL), PEG-PCL-PDEA (asymmetric), and PEG-SSPCL for facile loading and efficient intracellular delivery of proteins (Scheme 1). These multifunctional polymersomes were designed by following reasons: (i) they might exhibit superior protein loading due to presence of active interactions between proteins and PDEA chains inside the polymersomes, as reported for chimaeric PEG-PCL-PDEA polymersomes;9 (ii) Gal-conjugated micelles could efficiently target to asialoglycoprotein receptor (ASGP-R) overexpressing hepatoma cells;40−42 and (iii) they might quickly collapse in the cytoplasm and nuclei of cancer cells due to reductive cleavage of disulfide bonds that would cause efficient intracellular protein release. We reported previously that reduction-sensitive PEG-SS-PCL micelles mediated significantly enhanced intracellular release of doxorubicin.43 Remarkably, our results showed that both cytochrome C (CC) and granzyme B

(GrB)-loaded Gal-decorated polymersomes exhibited apparent targetability and potent antitumor activity to HepG2 cells. CC and GrB are intracellular proteins that are able to provoke effective cell apoptosis.5,44,45 Here, preparation of reductionsensitive chimaeric biodegradable polymersomes, loading of proteins, reduction-triggered protein release, hepatoma-targeting, and in vitro antitumor activity of protein-loaded polymersomes were investigated.

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EXPERIMENTAL SECTION

Materials. Methoxy poly(ethylene glycol) (PEG, Mn = 5.0 kg/mol, Fluka) was dried by azeotropic distillation from anhydrous toluene. The asymmetric triblock copolymer poly(ethylene glycol)-b-poly(ecaprolactone)-b-poly(2-(diethylamino)ethyl methacrylate) (PEGPCL-PDEA) (Mn = 5.0−18.2−2.7 kg/mol, PDI = 1.6), galactose functionalized targeting polymer Gal-PEG-PCL (Mn = 8.0−17.8 kg/ mol, PDI = 1.6), and PEG-SS-Py (Mn = 5.0 kg/mol) were synthesized according to our previous reports.9,40,43 ε-Caprolactone (ε-CL, 99%, Alfa Aesar) was dried over CaH2 and distilled under reduced pressure prior to use. Tetrahydrofuran (THF) was dried by refluxing over sodium wire and distilled prior to use. Dichloromethane (DCM), N,Ndimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were dried by refluxing over CaH2 and distilled before use. PEG 550 (Mn = 550 g/mol), dithiothreitol (DTT, 99%, Merck), cytochrome c from equine heart (CC, Sigma), recombinant human granzyme B (Biovision), bovine serum albumin V fraction (>98%, Roche), ovalbumin (Sigma), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium salt (ABTS, Amresco), Nile red (99%, Sigma), and fluorescein isothiocyanate (95%, Fluka) were used without further purification. Annexin V-FITC/propidium iodide (PI, KeyGEN Tech.) was used according to the supplier’s instruction. Characterization. 1H NMR spectra were recorded on a Unity Inova 400 spectrometer operating at 400 MHz using CDCl3 as a solvent. The chemical shifts were calibrated against residual solvent signals of CDCl3. The molecular weight and polydispersity of copolymers were determined by a Waters 1515 gel permeation chromatograph (GPC) instrument equipped with two linear PLgel columns (Mixed-C) following a guard column and a differential refractive-index detector. The measurements were performed using THF as the eluent at a flow rate of 1.0 mL/min at 30 °C and a series of narrow polystyrene standards for the calibration of the columns. The size of polymersomes was determined using dynamic light scattering 2874

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(DLS). Measurements were carried out at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments equipped with a 633 nm He−Ne laser using backscattering detection. The zeta potential of the polymersomes was determined with a Zetasizer Nano-ZS from Malvern Instruments. Transmission electron microscopy (TEM) was performed using a Tecnai G220 TEM operated at an accelerating voltage of 200 kV. The samples were prepared by dropping 10 mL of 0.1 mg/mL polymersome suspension on the copper grid followed by staining with phosphotungstic acid (1 wt %). The fluorescence images of polymersomes and cellular uptake were taken on confocal laser scanning microscope (TCS SP5) and inverted fluorescence microscope (Ti-S), respectively. Synthesis of PEG-SS-PCL. PEG-SS-PCL was prepared by ringopening polymerization of ε-CL using PEG-SS-OH as a macroinitiator and Sn(Oct)2 as a catalyst (Scheme S1). PEG-SS-OH was synthesized by treating PEG-SS-Py with 2-mercaptoethanol. Typically, under a nitrogen atmosphere to a solution of PEG-SS-Py (0.149 g, 0.0287 mmol) in 4.1 mL of water was added 50 μL of HCl solution (0.62 M) to adjust the pH to about 2.5. After complete dissolution of PEG-SSPy, 2-mercaptoethanol (5 μL, 5.6 mg, 0.0719 mmol) was added. The reaction was allowed to proceed under stirring for 48 h at 25 °C. The final product PEG-SS-OH was isolated by dialysis (MWCO 1000) against deionized water for 48 h followed by freeze-drying for 48 h. Yield: 87%. Then, under a nitrogen atmosphere, to a Schlenk vessel equipped with magnetic stirring bar, PEG-SS-OH (0.0625 g, 0.0121 mmol), Sn(Oct)2 (3 mg, 6.03 × 10−3 mmol), ε-CL (0.245 g, 2.115 mmol), and toluene (2 mL) were charged. The vessel was sealed and immersed in an oil bath thermostated at 100 °C. The polymerization was allowed to proceed under stirring for 48 h. The reaction was terminated by adding excess HCl. The resulting PEG-SS-PCL was isolated by precipitation in cold diethyl ether, filtration, and drying in vacuo. Yield: 92.8%. Mn (GPC) = 23.5 kg/mol, PDI (GPC) = 1.8. Preparation of Chimaeric Polymersomes. Reduction-sensitive chimaeric polymersomes were prepared by charging PEG-SS-PCL and PEG-PCL-PDEA copolymers at predetermined weight ratios (total weight 1.0 mg) as well as PEG 550 (10 μL) into a 1.5 mL tube. PEG550 is an excipient that helps to solubilize the copolymers. The mixture was heated with stirring at 95 °C for 15 min and then cooled to room temperature for 20 min. 10 μL of MES (20 mM, pH 5.3) was added. The mixture was stirred at 60 °C for 30 min and ultrasonicated for 5 min. Then, 20, 70, and 900 μL of MES (20 mM, pH 5.3) were added in that order with 5 min ultrasonication following each addition. The dispersion was stirred at 40 °C for 12 h and then dialyzed against the same MES twice and PB (20 mM, pH 7.4) twice for 12 h (MWCO 3500 Da). The obtained polymersomes were denoted as CP(SSxx), wherein xx represents the weight percentage (wt %) of PEG-SS-PCL. In a similar way, Gal-decorated reduction-sensitive chimaeric polymersomes were prepared from PEG-SS-PCL, PEG-PCL-PDEA, and GalPEG-PCL copolymers and denoted as Galyy-CP(SSxx), wherein xx and yy represent the weight percentage (wt %) of PEG-SS-PCL and Gal-PEG-PCL, respectively. The size, size distribution, and the zeta potential of chimaeric polymersomes were determined by using a Malvern Zetasizer NanoZS particle analyzer and its software, Dispersion Technology Software (DTS). The critical aggregation concentration (CAC) was determined using pyrene as a fluorescence probe with polymer concentrations ranging from 1 × 10−5 to 5 × 10−2 mg/mL and a fixed pyrene concentration of 0.6 μM, as described in our previous report.9 The polymersomal structure was studied using TEM and CLSM following loading with Nile red (hydrophobic) and FITC-CC (hydrophilic). Loading of Proteins into Polymersomes. Protein-loaded polymersomes were prepared in a similar way as the above description. For example, PEG-SS-PCL and PEG-PCL-PDEA copolymers at predetermined weight ratios (total weight 1.0 mg) and PEG 550 (10 μL) were changed into a 1.5 mL tube. The mixture was heated with stirring at 95 °C for 15 min and then cooled to room temperature for 20 min. 10 μL of protein solutions (10 mg/mL) in MES (pH 5.3, 20 mM) was applied (theoretical protein loading content = 9.1 wt %). The mixture was stirred at 60 °C for 30 min and ultrasonicated for 5

min. Then, 20, 70, and 900 μL of MES (20 mM, pH 5.3) were added in that order with 5 min ultrasonication following each addition. The dispersion was stirred at 40 °C for 12 h. The free proteins were removed by dialysis (MWCO 350 kDa) against MES (pH 5.3, 20 mM) for 10 h at 20 °C with at least 4 times change of media. Here, polymersomes were loaded with different proteins such as cytochrome c (CC), FITC-BSA, FITC-CC, and FITC-OVA. The control experiments on free proteins showed that this purification procedure is sufficient to remove free proteins if present. The loading of granzyme B (GrB) was carried out in a similar way except that 0.1 wt % GrB was loaded. To determine protein loading contents (PLC), protein-loaded polymersomes were supplemented with 3 times its volume of DMSO for 2 h to disrupt polymersomes and to release proteins. PLC and protein loading efficiency (PLE) for FITC-BSA, FITC-CC, and FITCOVA proteins were determined by fluorometry based on a calibration curve established with known concentrations of corresponding FITClabeled proteins in DMSO/water (3/1, v/v). PLC and PLE for CC were determined by UV−vis spectrometry using BCA protein assay kits (Pierce) according to supplier’s information. PLC and PLE were calculated according to the following formulas:

PLC (wt %) =

PLE (%) =

weight of loaded protein × 100% total weight of polymer and protein

weight of loaded protein × 100% weight of protein in feed

In Vitro Protein Release. The release of FITC-BSA or FITC-CC from polymersomes was investigated using a dialysis method (MWCO 350 kDa) at 37 °C with 0.6 mL of protein-loaded polymersome suspensions against 25 mL of PB (pH 7.4, 20 mM) with or without 10 mM DTT. At desired time intervals, 6 mL of release media was taken out and replenished with an equal volume of fresh media. The amounts of released proteins as well as proteins remaining in the dialysis tube were determined by fluorescence measurements (FLS920, excitation at 492 nm, emission from 492 to 690 nm). The release experiments were conducted in triplicate. Evaluation of Enzymatic Activity of CC Released from Polymersomes. The electron transfer activity of CC was measured by examining the catalytic conversion of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).46 CC-loaded polymersomes were placed into a dialysis tube (MWCO 350 kDa) and incubated in PB buffer (pH 7.4, 20 mM) containing 10 mM DTT overnight. CC released in the medium was quantified using BCA protein assay (Pierce) and then diluted by PB to a final concentration of 4.0 μg/mL. Subsequently, 10 μL of hydrogen peroxide solution (45 mM) and 100 μL of ABTS solution (1.0 mg/mL) in PB were added. The absorbance at 410 nm of the oxidized product was monitored every 20 s for 4 min. The same concentration of native CC was used as a control. Flow Cytometry Studies. MCF-7 or HepG2 cells were plated in a 24-well plate (5 × 104 cells/well) under a 5% CO2 atmosphere at 37 °C using DMEM medium supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL) for 24 h. The cells were treated with a predetermined amount of CC-loaded chimaeric polymersomes or free CC (CC dosage: 40 μg/mL) under a 5% CO2 atmosphere at 37 °C for 48 h. To quantify apoptotic cells, an Annexin V-FITC kit was used as described by the manufacturer (KenGEN, China). Briefly, cells were digested with EDTA-free trypsin, washed twice with cold PBS, and resuspended in binding buffer at a concentration of 1 × 105 cells/mL. Then the cells were stained with 5 μL of Annexin V-FITC solution and 5 μL of propidium iodide (PI) solution for 15 min at room temperature in the dark. At the end of incubation, 400 μL of binding buffer was added, and the cells were analyzed immediately using flow cytometry (BD FACSCalibur, Mountain View, CA). Evaluation of Apoptotic Activity of Protein-Loaded Polymersomes by MTT Assays. The antitumor activity of CC or GrBloaded hepatoma-targeting chimaeric polymersomes in asialoglycoprotein receptor (ASGP-R) overexpressing HepG2 cells was evaluated 2875

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by MTT assays. HepG2 cells pretreated with free LBA (1 mg/mL), and MCF-7 cells (low ASGP-R expression) were used as negative controls. The cells were plated in a 96-well plate (5 × 103 cells/well) for 24 h. The medium was aspirated and replaced by 90 μL of fresh medium. 10 μL of CC or GrB-loaded Gal20-CP(SS40) and CP(SS40) were added to yield a final CC concentration of 40 μg/mL and GrB concentrations ranging 1.0 × 10−4 to 0.4 μg/mL. The cells were then cultured in DMEM medium at 37 °C under a 5% CO2 atmosphere for 4 h. The medium was aspirated and replaced by 100 μL of fresh medium. The cells were cultured for another 68 and 116 h for GrB and CC-loaded polymersomes, respectively. Then, 10 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution (5 mg/mL) was added. The cells were incubated for an additional 4 h. The medium was aspirated, the MTT-formazan generated by live cells was dissolved in 150 μL of DMSO, and the absorbance at a wavelength of 492 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 492 nm with control wells containing only cell culture medium. Data are presented as average ± SD (n = 4). For inhibition experiments, HepG2 cells were pretreated with free LBA (1 mg/mL) for 4 h to block the ASGP-R receptors on the cell surface, the media were aspirated and replaced by fresh cell culture media, and then protein-loaded polymersomes were added. To study the effect of the galactose content in polymersomes on their targetability, antitumor activity of CC-loaded CP(SS40), Gal10CP(SS40), Gal20-CP(SS40), Gal30-CP(SS40), and Gal40-CP(SS40) to HepG2 cells was evaluated at CC concentrations of 0.1−40 μg/mL. The cytotoxicity of CP(SS50) and Gal20-CP(SS40) to HepG2, MCF-7, and HeLa cells was determined in a similar way at polymer concentrations of 0.5, 0.75, and 1.0 mg/mL. Cellular Uptake and Intracellular Protein Release. MCF-7 or HepG2 cells were plated on microscope slides in a 24-well plate (5 × 104 cells/well) under a 5% CO2 atmosphere at 37 °C using DMEM medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and antibiotics penicillin (100 IU/mL) and streptomycin (100 μg/ mL) for 24 h. To investigate influences of disulfide contents on the intracellular protein release, FITC-CC-loaded chimaeric polymersomes containing 0, 30, 50, and 70 wt % PEG-SS-PCL were used to incubate with MCF-7 cells for 24 h at 37 °C (FITC-CC dosage: 40 μg/mL). Free CC was used as a control. To evaluate hepatoma targetability of Gal-decorated polymersomes, HepG2 cells or LBA pretreated HepG2 cells were incubated for 4 h with FITC-CC-loaded Gal20-CP(SS40), FITC-CC-loaded CP(SS40), or FITC-CC (FITCCC dosage = 40 μg/mL). The medium was aspirated and replaced by fresh medium. The cells were cultured for another 2 or 20 h, and the medium was removed. The cells on microscope slides were washed three times with PBS and subsequently fixed with 4% formaldehyde for 20 min followed by three times wash with PBS. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 20 min and washed three times with PBS. Fluorescence images were obtained using a confocal microscope (TCS SP5).

Gal-PEG-PCL was equipped with a longer PEG chain than that in PEG-PCL-PDEA and PEG-SS-PCL copolymers (8.0 vs 5.0 kg/mol) to fully expose Gal ligands at the outer surface of polymersomes. Formation of Reduction-Sensitive Chimaeric Polymersomes. Chimaeric polymersomes were readily prepared using the direct hydration method from asymmetric PEG-PCLPDEA triblock copolymer and PEG-SS-PCL diblock copolymer at prescribed weight ratios (Table 1). Thus-obtained polymerTable 1. Characteristics of Reduction-Sensitive Chimaeric Polymersomes Based on PEG-SS-PCL and Asymmetric PEG-PCL-PDEA Copolymers entry

polymersomes

sizea (nm)

1 2 3 4 5 6

CP(SS90) CP(SS70) CP(SS50) CP(SS30) CP(SS0) PEG-SS-PCL

199.2 186.4 144.0 103.3 95.5 210.0

± ± ± ± ± ±

4.0 1.8 3.9 2.1 1.7 2.2

PDIa 0.21 0.17 0.09 0.13 0.10 0.23

ζb (mV)

CAC (mg/L)

± ± ± ± ± ±

1.18 1.95 1.83 2.00 2.22 0.11

0.65 0.73 0.68 0.92 0.98 0.40

0.2 0.3 0.1 0.4 0.2 0.2

a

Determined by DLS in PB (pH 7.4, 20 mM) at a polymer concentration of 1.0 mg/mL. bDetermined by zeta potential measurements in PB (pH 7.4, 20 mM).

somes were denoted as CP(SSxx), wherein xx represents weight percentage (wt %) of PEG-SS-PCL. Dynamic light scattering (DLS) showed that all polymersomes had a monodisperse distribution (Figure 1A). The average sizes of chimaeric polymersomes increased from 99.5 to 199.2 nm with increasing PEG-SS-PCL contents from 0 to 90 wt % (Table 1). We found that asymmetric triblock copolymer tends to form small-sized polymersomes likely due to its favored formation of curvature structures.9 TEM micrograph revealed that these

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RESULTS AND DISCUSSION The aim of present study was set to develop multifunctional biodegradable polymersomes for efficient hepatoma-targeting protein delivery, for which novel reduction-sensitive chimaeric polymersomes were designed based on following three components: (i) asymmetric PEG-PCL-PDEA triblock copolymer (Mn = 5.0−18.2−2.7 kg/mol) to facilitate formation of chimaeric polymersomes as well as efficient loading of proteins into the watery interior of polymersomes via ionic interactions;9 (ii) PEG-SS-PCL diblock copolymer (Mn = 5.0−17.6 kg/mol) to render fast protein release in the cytosol and cell nucleus owing to reductive cleavage of disulfide bonds;43 and (iii) galactose-PEG-PCL diblock copolymer (Gal-PEG-PCL, Mn = 8.0−17.8 kg/mol) for active targeting to asialoglycoprotein receptor (ASGP-R) overexpressing hepatoma cells.40 Notably,

Figure 1. Characteristics of reduction-sensitive chimaeric polymersomes based on PEG-SS-PCL and asymmetric PEG-PCL-PDEA copolymers: (A) size distribution profiles of CP(SS0), CP(SS30), CP(SS50),CP(SS70), CP(SS90), and PEG-SS-PCL polymersomes determined by DLS; (B) TEM micrograph of CP(SS50); (C) change of CP(SS50) sizes in response to 10 mM DTT in PB (pH 7.4, 20 mM); and (D) cytotoxicity of CP(SS50) toward HeLa and MCF-7 cells following 24 h incubation determined by MTT assays. Data are shown as mean ± SD (n = 4). 2876

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Table 2. Loading of Proteins into Reduction-Sensitive Chimaeric Polymersomes of PEG-SS-PCL and Asymmetric PEG-PCLPDEA Copolymersa entry

proteins

polymersomes

sizeb (nm)

1 2 3 4 5 6 7 8 9

FITC-BSA

CP(SS70) CP(SS50) CP(SS30) CP(SS0) PEG-SS-PCL CP(SS50) CP(SS30) CP(SS50) CP(SS30)

179.0 141.9 125.8 95.5 195.6 146.5 129.6 152.9 117.8

FITC-CC FITC-OVA

± ± ± ± ± ± ± ± ±

3 2 3 1 5 3 3 2 5

PDIb

ζc (mV)

PLCd (wt %)

PLEd (%)

0.17 0.14 0.18 0.15 0.20 0.19 0.15 0.22 0.18

0.58 1.05 0.98 1.03 0.03 1.02 0.95 0.10 0.16

6.1 6.2 7.0 7.8 3.2 3.9 6.1 6.6 7.0

67.2 70.3 79.7 89.4 35.6 43.6 66.7 72.7 76.8

a

Polymersomes were prepared in PB (pH 7.4, 20 mM) at a polymer concentration of 1.0 mg/mL and theoretical protein loading content (PLC) of 9.1 wt % (n = 5). bDetermined by DLS in PB (pH 7.4, 20 mM). cDetermined by zeta potential measurements in PB (pH 7.4, 20 mM). dDetermined by fluorometry.

Table 3. Characteristics of CC-Loaded Gal-Decorated Reduction-Sensitive Chimaeric Polymersomes Based on 40 wt % PEGSS-PCLa entry

polymersomes

1 2 3 4 5

Gal10-CP(SS40) Gal20-CP(SS40) Gal30-CP(SS40) Gal40-CP(SS40) CP(SS40)

sizeb (nm) 145.8 155.6 160.4 164.8 132.4

± ± ± ± ±

3 4 6 3 4

PDIb

ζc (mV)

PLCd (wt %)

PLEd (%)

IC50 (μM)

0.20 0.18 0.22 0.20 0.21

1.03 1.25 0.89 1.25 1.16

4.8 3.8 3.2 2.7 5.0

53.1 42.2 34.8 30.3 55.3

1.66 0.96 0.50

a Polymersomes were prepared in PB (pH 7.4, 20 mM) at a polymer concentration of 1.0 mg/mL and theoretical CC loading content of 9.1 wt % (n = 5). bDetermined by DLS in PB (pH 7.4, 20 mM). cDetermined by zeta potential measurements in PB (pH 7.4, 20 mM). dDetermined by fluorometry.

0.19) with increasing Gal-PEG-PCL contents from 10 to 40 wt % (PEG-PCL-PDEA contents decreasing accordingly from 50 to 20 wt %). Loading and Reduction-Triggered Release of Model Proteins. Proteins could be readily loaded into chimaeric polymersomes via the direct hydration method. The loading of FITC-labeled proteins such as bovine serum albumin (BSA), ovalbumin (OVA), and cytochrome c (CC) was studied. The results showed that protein loading efficiencies (PLE) were proportional to the PEG-PCL-PDEA contents in the polymersomes (Table 2). For example, at a theoretical protein loading content (PLC) of 9.1 wt %, PLE of FITC-BSA increased from 35.6% to 89.4% with increasing PEG-PCL-PDEA contents from 0 to 100%. Notably, CP(SS30) and CP(SS50) exhibited decent loading of FITC-BSA, FITC-OVA, and FITC-CC. The average sizes of FITC-BSA-loaded polymersomes decreased from 195.6 to 95.5 nm (PDI = 0.14−0.22) with increasing PEG-PCLPDEA contents from 0 to 100%, while zeta potentials were all close to neutral. The protein-loaded CP(SS30) and CP(SS50) displayed good sizes of ca. 120−150 nm. In the following, we studied loading of CC into Galdecorated CP(SS40). The results showed that with increasing Gal-PEG-PCL from 0 to 40 wt % (i.e., decreasing PEG-PCLPDEA from 60 to 20 wt %), PLE decreased from 55.3% to 30.3%, and polymersome sizes increased from 132.4 to 164.8 nm (Table 3), further confirming that PEG-PCL-PDEA plays a critical role in determining polymersome sizes and protein loading capacity. The in vitro release behavior of protein-loaded reductionsensitive chimaeric polymersomes was investigated at pH 7.4 and 37 °C in the presence or absence of 10 mM dithiothreitol (DTT). Interestingly, under physiological conditions, release of both FITC-BSA and FITC−CC was inhibited by incorporating

polymersomes had a vesicular structure and spherical morphology (Figure 1B). The vesicular structure was further confirmed by confocal microscopy observations, in which fluorescence of hydrophilic FITC−cytochrome c (FITC-CC) and hydrophobic Nile red colocalized and moreover FITC-CC appeared to reside inside of polymersomes (Figure S2). Notably, these chimaeric polymersomes had low critical aggregation concentrations (CAC) of 1.18−2.22 mg/L and zeta potentials close to neutral (Table 1), supporting that PDEA is preferentially located inside of polymersomes. They remained stable for at least 3 days under physiological conditions (PBS, pH 7.4, 20 mM, 150 mM NaCl). In contrast to rapid swelling and collapse of PEG-SS-PCL polymersomes in response to 10 mM DTT in PB (pH 7.4, 20 mM) (Figure S3), little size change was observed for chimaeric polymersomes prepared from PEG-PCL and PEG-SS-PCL in 24 h under otherwise the same conditions (Figure 1C), suggesting that these block copolymers mix well and form one homogeneous vesicle population. This result is in accordance with shellsheddable micelles based on PEG-PCL and PEG-SS-PCL, in which micelles maintain adequate colloidal stability even after shedding off ca. 90% of PEG shells.47 MTT assays showed that these polymersomes were essentially nontoxic to HeLa and MCF-7 cells (>87% cell viabilities) up to a tested concentration of 1.0 mg/mL (Figure 1D). In a similar way, Gal-decorated reduction-sensitive chimaeric polymersomes were prepared from PEG-PCL-PDEA, PEG-SSPCL, and Gal-PEG-PCL copolymers. The PEG-SS-PCL content was fixed at 40 wt % while Gal-PEG-PCL contents varied from 10 to 40 wt %. The resulting polymersomes were denoted as Galyy-CP(SS40), wherein yy represents weight percentage (wt %) of Gal-PEG-PCL. The average sizes of polymersomes increased from 128.6 to 160.2 nm (PDI 0.11− 2877

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PEG-SS-PCL into chimaeric polymersomes (Figure 2). For instance, ca. 57, 25, 18, and 12% of FITC-BSA were released in

12 h from CP(SS0), CP(SS30), CP(SS50), and CP(SS70), respectively (Figure 2A). The fast protein release from CP(SS0) is likely due to partial segregation of proteins and PDEA in the polymersome membrane.9 The presence of PEGSS-PCL would favor short PDEA and thereby proteins to locate inside the polymersomes, thus reducing protein release. However, protein release from reduction-sensitive chimaeric polymersomes was significantly accelerated in the presence of 10 mM DTT under otherwise the same conditions, in which the higher the amount of PEG-SS-PCL in the polymersomes, the faster the protein release. For example, 58.5, 63.2, 77.2, and 88.4% of FITC-BSA were released from CP(SS0), CP(SS30), CP(SS50), and CP(SS70) polymersomes in 12 h (Figure 2A). Similar phenomena were also observed for FITC-CC release (Figure 2B). The presence of 10 mM DTT while having little influence on protein release from CP(SS0) markedly enhanced protein release from CP(SS30), CP(SS50), and CP(SS70), supporting that protein is released from reduction-sensitive chimaeric polymersomes in response to an intracellularmimicking reductive environment and protein release rate can be regulated by PEG-SS-PCL content. Therefore, by combining PEG-SS-PCL and asymmetric PEG-PCL-PDEA copolymers, we can obtain chimaeric polymersomes with decent protein loading and reduction-triggered protein release behavior. Cellular Uptake and Apoptotic Activity of CC-Loaded Chimaeric Polymersomes. It is important that released proteins maintain their biological activity. Here, we chose CC as a model protein. The enzymatic activity and apoptotic activity of CC-loaded chimaeric polymersomes were evaluated by ABTS assay and flow cytometry, respectively. The results showed that there is no significant difference in UV absorption of oxidized ABTS catalyzed either by CC released from chimaeric polymersomes or by free CC (Figure S4), implying that released CC maintains its enzymatic activity.9,46 It is known that CC also plays a significant role in programmed cell death.48 Once released into the cytoplasm from mitochondria upon substantial cell damage, CC initiates a molecular cascade leading to proteolytic cleavage of cellular proteins, as part of

Figure 2. In vitro protein release profiles from reduction-sensitive chimaeric polymersomes in PB (20 mM, pH 7.4) at 37 °C in the presence or absence of 10 mM DTT (n = 3). PEG-SS-PCL polymersomes were used as a control. The polymersome concentration was fixed at 1.0 mg/mL. (A) FITC-BSA and (B) FITC-CC.

Figure 3. Contour diagram of Annexin V-FITC/PI flow cytometry of MCF-7 cells following 48 h incubation with CC-loaded reduction-sensitive chimaeric polymersomes. CC-loaded PEG-SS-PCL polymersomes, free CC, empty CP(SS50), and blank cells were used as controls. CC concentration was fixed at 40 μg/mL. 2878

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Figure 4. CLSM images of HepG2 cells incubated with FITC-CC-loaded Gal20-CP(SS40) for 6 h (A) or 24 h (B). FITC-CC concentration was set at 40 μg/mL. Control experiments: (C) FITC-CC-loaded CP(SS40), 24 h; (D) free FITC-CC, 24 h; (E) HepG2 cells were pretreated with LBA (1 mg/mL) followed by incubation with FITC-CC-loaded Gal20-CP(SS40) for 24 h. For each panel, images from left to right were nuclei stained by DAPI (blue), FITC-CC fluorescence (green), overlays of the above two images. The scale bar represents 20 μm.

reduction-triggered protein release is of utmost importance for CC to exert the apoptotic effect. The cellular uptake and intracellular protein release of FITCCC-loaded polymersomes were studied in MCF-7 cells by fluorescence microscopy. Interestingly, the results showed clearly that FITC-CC fluorescence intensity inside cells correlated well with PEG-SS-PCL content in polymersomes, in which cells treated with FITC-CC-loaded CP(SS70) showed significantly stronger fluorescence than those with FITC-CCloaded CP(SS50) and CP(SS30) (Figure S5). In contrast, little fluorescence was detected in cells incubated with CP(SS0) and free CC under otherwise the same conditions. These observations are in accordance with apoptotic activity of CCloaded chimaeric polymersomes (Figure 3).

programmed cell death. It is reported that intracellularly microinjected CC and CC-loaded nanoparticles or polymersomes could provoke cell apoptosis.46,49 The apoptotic activity of CC-loaded reduction-sensitive chimaeric polymersomes was investigated in MCF-7 cells by flow cytometry using annexin VFITC/propidium iodide (PI) staining at a CC dosage of 40 μg/ mL. The results revealed that CC-loaded reduction-sensitive chimaeric polymersomes induced obviously higher apoptotic activity than free CC, in which CC-loaded CP(SS0), CP(SS30), CP(SS50), CP(SS70), and PEG-SS-PCL polymersomes caused ca. 5.5, 8.0, 11.0, 18.2, and 22.4% of cell death, respectively (Figure 3). In contrast, minimal cell death (4.5%) was observed for cells treated with free CC likely due to poor cellular uptake. The higher apoptotic activity observed for CC-loaded chimaeric polymersomes with a higher PEG-SS-PCL content signifies that 2879

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Hepatoma-Targeted Delivery of CC Using Gal-Decorated Reduction-Sensitive Chimaeric Polymersomes. To further improve cellular uptake and antitumor activity of CCloaded reduction-sensitive chimaeric polymersomes, polymersome surfaces were decorated with β-D-galactose (Gal) by incorporating Gal-PEG-PCL. It is known that Gal ligand exhibits high specificity to asialoglycoprotein receptor (ASGPR)-overexpressing hepatoma cells including HepG2 cells.50 Notably, confocal microscopy observations displayed that Gal20-CP(SS40) delivered and released FITC-CC into HepG2 cells in 6 h and FITC fluorescence inside cells was further enhanced after 24 h, which was significantly stronger than that in cells treated with nontargeting CP(SS40) or free CC under otherwise the same conditions (Figure 4). The inhibition experiments showed that FITC fluorescence in HepG2 cells was reduced by pretreating the cells with lactobionic acid (LBA, 1 mg/mL) prior to incubating with FITC-CC-loaded Gal20-CP(SS40). Figure S6 shows that FITC fluorescence intensity in HepG2 cells treated with FITC-CCloaded Gal20-CP(SS40) was approximately 3 times higher than that with the nontargeting counterpart. These results confirm that Gal20-CP(SS40) was taken up by HepG2 cells via a receptor-mediated mechanism. The antitumor activity of CC-loaded Gal20-CP(SS40) was investigated using MTT assays. The cells were incubated with CC-loaded polymersomes or free CC (40 μg/mL) for 4 h, media was aspirated and replenished with fresh culture media, and cells were further cultured for another 116 h. The results showed that CC-loaded Gal20-CP(SS40) was significantly more potent toward HepG2 cells than the nontargeting CP(SS40) counterparts, in which ca. 50.0 and 80.1% cell viabilities were observed for CC-loaded Gal20-CP(SS40) and CP(SS40), respectively (Figure 5A). In contrast, CC-loaded Gal20-CP(SS40) caused similar apoptotic effect (cell viability = ca. 82.2%) to CC-loaded CP(SS40) in MCF-7 cells (negative control) under otherwise identical conditions. Moreover, the apoptotic activity of CC-loaded Gal20-CP(SS40) was significantly reduced by pretreating HepG2 cells with LBA. The pretreatment of HepG2 cells with LBA, on the other hand, had little influence on apoptotic potency of CC-loaded CP(SS40) or free CC. In all cases, free CC brought about negligible cell death. These results point out that Gal20-CP(SS40) possesses apparent targetability to HepG2 cells and can efficiently deliver and release apoptotic proteins into target cells inducing effective cell apoptosis. Notably, Gal20-CP(SS40) was practically nontoxic to both HepG2 and MCF-7 cells (cell viabilities >90%) up to a tested concentration of 1.0 mg/mL (Figure S7). We further studied the influences of Gal densities on the apoptotic activity of CC-loaded CP(SS40). Interestingly, MTT results demonstrated that reduction-sensitive chimaeric polymersomes with higher Gal contents provoked progressively higher level of HepG2 cell apoptosis (Figure 5B). For example, CC-loaded Gal10-CP(SS40), Gal20-CP(SS40), Gal30-CP(SS40), and Gal40-CP(SS40) caused about 39.6, 50.3, 57.5, and 68.6% of cell apoptosis, respectively, at a CC dosage of 40 μg/mL in 120 h, which were all significantly higher than 18.7% of cell apoptosis observed for nontargeting CP(SS40). The half-maximal inhibitory concentration (IC50) values of CCloaded Gal20-CP(SS40), Gal30-CP(SS40), and Gal40-CP(SS40) were determined to be ca. 1.66, 0.96, and 0.50 μM CC, respectively (Table 3). Hepatoma-Targeted Delivery of Granzyme B. In order to further demonstrate the applicability of Gal-decorated

Figure 5. (A) Antitumor activity of CC-loaded Gal20-CP(SS40), CCloaded CP(SS40), and free CC (CC dosage = 40 μg/mL) to HepG2 cells. HepG2 cells pretreated with LBA (1 mg/mL) and MCF-7 cells were used as controls. (B) Influences of Gal contents on the antitumor activity of CP(SS40) toward HepG2 cells (CC dosage ranging from 0.1 to 40 μg/mL). The cells were incubated with CC-loaded polymersomes or free CC for 4 h, media was removed and replenished with fresh culture media, and cells were further cultured for another 116 h. Data are shown as mean ± SD (n = 4) (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001).

reduction-sensitive chimaeric polymersomes for protein therapy, granzyme B (GrB), a highly potent apoptosis mediator, was loaded into Gal20-CP(SS40). GrB induces cell death by a mechanism primarily used by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, which are the two main types of cytotoxic effector cells of the immune system.44 After recognition of the target cells, the engaged CTLs and NK cells secrete GrB along with other granule proteins including perforin. With the help of the pore-forming perforin, GrB translocates into the cytosol of target cells to cleave, activate, or inactivate multiple protein substrates, resulting in apoptosis of target cells.51 Microinjected GrB was reported to effectively kill human tumor cells that are otherwise resistant to many cytotoxic drugs.45 Gal20-CP(SS40) following loading with 0.1 wt % GrB had an average size of 150.2 nm and a PDI of 0.17. MTT assays displayed that GrB-loaded Gal20CP(SS40) possessed superior antitumor activity to HepG2 cells, in which a high cell apoptosis (ca. 62.5%) was achieved in 72 h at a low GrB concentration of 0.4 μg/mL (Figure 6). The IC50 was calculated to be ca. 0.069 μg/mL (ca. 2.7 nM), which is comparable to that of microinjected GrB into MCF-7 cells 2880

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presence of active interactions between protein and PDEA chains located inside the polymersomes; (iii) they show apparent targetability to hepatoma cells and surface density of galactose ligand can conveniently be tuned by Gal-PEG-PCL contents; and (iv) they rapidly release proteins in response to the reductive environment in the cytosol and nucleus of cancer cells. Interestingly, our results have shown that cytochrome c and granzyme B-loaded Gal-decorated reduction-sensitive chimaeric polymersomes cause great antitumor activity toward HepG2 cells. In particular, granzyme B-loaded targeting polymersomes might be applied as hepatoma specific “artificial killer cells”. These reduction-sensitive chimaeric polymersomes have provided a versatile and multifunctional platform for targeted protein therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 6. Antitumor activity of GrB-loaded Gal20-CP(SS40), GrBloaded CP(SS40), and free GrB (GrB dosages ranging from 1.0 × 10−4 to 0.4 μg/mL) to HepG2 cells. HepG2 cells pretreated with LBA (1 mg/mL) and MCF-7 cells were used as controls. The cells were incubated with GrB-loaded polymersomes or free GrB for 4 h, media was removed and replenished with fresh culture media, and cells were further cultured for another 68 h. Data are shown as mean ± SD (n = 4) (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001).

Synthesis and 1H NMR spectrum of PEG-SS-PCL; CLSM images of CP(SS50) loaded with FITC-CC and Nile red; oxidation of ABTS catalyzed by CC released from CP(SS50); fluorescence images of MCF-7 cells incubated with FITC-CCloaded polymersomes; fluorescence intensity from CLSM images of HepG2 cells incubated with FITC-CC-loaded Gal20-CP(SS40); and cytotoxicity of Gal20-CP(SS40) toward HepG2 and MCF-7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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(0.04 μg/mL GrB led to 40% apoptosis).45 Remarkably, GrBloaded Gal20-CP(SS40) had about 600-fold lower IC50 than CC-loaded Gal20-CP(SS40) in HepG2 cells. Moreover, GrBloaded Gal20-CP(SS40) was much more potent than chemotherapeutics, with IC50 several hundreds times lower than that reported for doxorubicin, paclitaxel, or docetaxel against HepG2 cells.42,52−54 In contrast, GrB-loaded nontargeting CP(SS40) and free GrB displayed significantly lower apoptotic activity with only about 31.5% and 20.2% cell apoptosis, respectively, under otherwise the same conditions (Figure 6). It should be noted that GrB-loaded Gal20-CP(SS40) caused modest apoptosis of MCF-7 cells (negative control), confirming that GrB-loaded Gal20-CP(SS40) is taken up by HepG2 cells via a receptor-mediated mechanism. The targetability of GrB-loaded Gal20-CP(SS40) to HepG2 cells was further supported by inhibition experiments, in which GrBloaded Gal20-CP(SS40) revealed greatly declined apoptotic activity to HepG2 cells pretreated with LBA. To the best of our knowledge, this represents a first report on design of nanosystems for delivery of GrB into target cancer cells. These granzyme B-loaded targeting polymersomes might be applied as hepatoma specific “artificial killer cells”. Hence, reduction-sensitive chimaeric polymersomes have appeared as a particularly versatile and multifunctional platform to efficiently chaperone therapeutic proteins into pathological cells, which holds a great potential in protein therapy.

AUTHOR INFORMATION

Corresponding Author

*Tel +86-512-65880098; e-mail [email protected] (F.M.), [email protected] (Z.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51003070, 51103093, 51173126, 51273137, and 51273139), the National Science Fund for Distinguished Young Scholars (NSFC 51225302), and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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CONCLUSIONS We have demonstrated that galactose-decorated reductionsensitive chimaeric polymersomes based on Gal-PEG-PCL, PEG-SS-PCL, and asymmetric PEG-PCL-PDEA copolymers actively deliver and release potent therapeutic proteins into hepatoma cells, resulting in efficient cell apoptosis. These multifunctional biodegradable polymersomes have several unique advantages: (i) they are readily prepared with small particle sizes of ca. 100−160 nm and without use of organic solvents; (ii) they have decent protein loading capacity due to 2881

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